The present invention relates to methods of using the three-dimensional quadrupole ion trap mass spectrometer ("ion trap") which was initially patented in 1960 by Paul, et al., (U.S. Pat. No. 2,939,952). In recent years use of the ion trap mass spectrometer has grown dramatically, in part due to its relatively low cost, ease of manufacture, and its unique ability to store ions over a large range of masses for relatively long periods of time. This latter feature makes the ion trap especially useful in isolating and manipulating individual ion species, as in a so-called tandem MS or "MS/MS" experiment where a "parent" ion species is isolated and fragmented or dissociated to create "daughter" ions which may then be identified using traditional ion trap detection methods or further fragmented to create granddaughter ions, etc. Nonetheless, there is a need to improve high mass resolution and reproducibility of results in ion traps. A major factor limiting the mass resolution and reproducibility is space charge which can alter the trapping conditions from one experiment to the next unless held at a constant level.
The quadrupole ion trap comprises a ring-shaped electrode and two end cap electrodes. Ideally, both the ring electrode and the end cap electrodes have hyperbolic surfaces that are coaxially aligned and symmetrically spaced. By placing a combination of AC and DC voltages (conventionally designated "V" and "U", respectively) on these electrodes, a quadrupole trapping field is created. A trapping field may be simply created by applying a fixed frequency (conventionally designated "f") AC voltage between the ring electrode and the end caps to create a quadrupole trapping field. The use of an additional DC voltage is optional, and in commercial embodiments of the ion trap no DC voltage is normally used. It is well known that by using an AC voltage of proper frequency and amplitude, a wide range of masses can be simultaneously trapped.
The mathematics of the quadrupole trapping field created by the ion trap are well known and were described in the original Paul, et al., patent. For a trap having a ring electrode of a given equatorial radius r.sub.0, with end cap electrodes displaced from the origin at the center of the trap along the axial line r=0 by a distance z.sub.0, and for given values of U, V and f, whether an ion of mass-to-charge ratio (m/e, also frequently designated m/z) will be trapped depends on the solution to the following two equations: ##EQU1## where .omega. is equal to 2 #f.
Solving these equations yields values of a.sub.z and q.sub.z for a given ion species having the selected m/e. If the point (a.sub.z,q.sub.z) maps inside the stability envelop, the ion will be trapped by the quadrupole field. If the point (a.sub.z,q.sub.z) falls outside the stability envelop, the ion will not be trapped and any such ions that are introduced within the ion trap will quickly move out of the trap. By changing the values of U, V or f one can affect the stability of a particular ion species. Note that from Eq. 1, when U=0, (i.e., when no DC voltage is applied to the trap), a.sub.z =0.
(It is common in the field to speak in abbreviated fashion in terms of the "mass" of ions, although it would be more precise to speak of the mass-to-charge ratio of ions, which is since that is what really affects the behavior of an ion is a trapping field. For convenience, this specification adopts the common practice, and generally uses the term "mass" as shorthand to mean mass-to-charge ratio.)
The typical method of using an ion trap consists of applying voltages to the trap electrodes to establish a trapping field which will retain ions over a wide mass range, introducing a sample into the ion trap, ionizing the sample, and then scanning the contents of the trap so that the ions stored in the trap are ejected and detected in order of increasing mass. Typically, ions are ejected through perforations in one of the end cap electrodes and are detected with an electron multiplier.
A number of methods exist for ionizing sample molecules. Most commonly, sample molecules are introduced into the trap and an electron beam is turned on, ionizing the sample within the trap volume. This is referred to as electron impact ionization or "EI". Alternatively, ions of a reagent compound can be created within or introduced into the ion trap to cause ionization of the sample due to interactions between the reagent ions and sample molecules. This technique is referred to as chemical ionization or "CI". Other methods of ionizing the sample, such as photoionization using a laser beam or other light source, are also known. For purposes of the present invention the specific ionization technique used to create ions is generally not important.
The various known ionization techniques all involve what will be referred to as "ionization parameters" that effect the number of ions created or introduced into the ion trap. In turn, the number of ions stored within the trap volume determines the space charge within the trap, since the space charge in the trap is a function of the overall ion population. Various ionization parameters may be used to control the number of ions introduced in the trap depending on the specific method of ion introduction. For example, when using EI, the number of ions created in the trap is a function of the intensity of the electron beam used to create the ions as well as the length of time the beam is turned on. Thus, both of these are ionization parameters as that term is used in the present specification, since the ion population in the trap can be controlled by varying the intensity of the beam or by varying the length of time the beam is turned on. Likewise, when using photoionization, both the length of time the light beam is turned on and the intensity of the beam are considered ionization parameters.
When using CI, the reaction time between the sample molecules and the reagent ions is an ionization parameter. It is noted that reagent ions are normally created within the ion trap by ionizing reagent molecules using an electron beam. In other words, the reagent ions are normally created by EI. In such a situation, the quantity of reagent ions created in the ion trap is dependent on the same ionization parameters described above, i.e., the length of time the electron beam is turned on and the intensity of the beam. When ionizing reagent ions, measures are normally taken to eliminate any sample ions simultaneously formed in the ion trap. According to the present invention, another method of creating reagent ions for a CI experiment is to allow initial precursor ions to react with a reagent gas to form the desired reagent ions. Thus, the reagent ions are themselves formed by chemical ionization.
While in most instances sample ions are created within the trap volume, in some instances ions may be created externally by any of the foregoing methods and transported into the ion trap using known ion transport means. In such instances, an electronic gating arrangement may be used to control the flow of ions into the trap, and the length of time the ion gate is "open" can be used to control the ion population introduced into the ion trap. Thus, this would also be considered an ionization parameter according to the present invention.
As described, there are a number of known methods for creating the ions that are trapped in an ion trap. For purposes of this specification, the terms "introduced" and "introducing," when used in connection with sample ions, are intended to cover all of the various methods. Thus, ions may be introduced into the ion trap either by formation within the trap volume, as by traditional in-trap EI or CI techniques, or by formation outside of the ion trap and transport into the trap volume.
Once the ions are formed and stored in the trap a number of techniques are available for isolating specific ions of interest, and for conducting so-called MS/MS experiments, sometimes called (MS).sup.n experiments. As noted, in MS/MS experiments an isolated ion or group of ions, called "parent" ions, are fragmented creating "daughter" ions, which may be detected themselves or fragmented to create "granddaughter" ions, etc. Techniques for isolating parent, daughter, etc., ions in an ion trap involve manipulating the trapping voltage(s) and/or using supplemental voltages as described in greater detail below. One particularly useful method of isolating an individual ion species in an ion trap is described in U.S. Pat. No. 5,198,665 (the '665 patent) issued to the present inventor and coassigned herewith. The disclosure of the '665 patent is hereby incorporated by reference.
Obtaining a mass spectrum generally involves scanning the trap so that ions are removed from the ion trap and detected. U.S. Pat. No. 4,540,884 to Stafford, et al., describes a technique for scanning one or more of the basic trapping parameters of the quadrupole trapping field, i.e., U, V or f, to sequentially cause trapped ions to become unstable and leave the trap. Unstable ions tend to leave in the axial direction and can be detected using a number of techniques, for example, as mentioned above, a electron multiplier or Faraday collector connected to standard electronic amplifier circuitry.
In the preferred method taught by the '884 patent, the DC voltage, U, is set at 0. As noted, from Eq. 1 when U=0, then a.sub.z =0 for all mass values. As can be seen from Eq. 2, the value of q.sub.z is directly proportional to V and inversely proportional to the mass of the particle. Likewise, the higher the value of V the higher the value of q.sub.z. In the preferred embodiment the scanning technique of the '884 patent is implemented by ramping the value of V. As V is increased positively, the value of q.sub.z for a particular mass increases to the point where it passes from a region of stability to one of instability. Consequently, the trajectories of ions of increasing mass to charge ratio become unstable sequentially, and are detected when they exit the ion trap.
According to another known method of scanning the contents of an ion trap, a supplemental AC voltage is applied across the end caps of the trap to create an oscillating dipole field supplemental to the quadrupole field. (Sometimes this combination of a quadrupole trapping field and a supplemental rf dipole field is referred to as a "combined field.") In this method, the supplemental AC voltage has a different frequency than the primary AC voltage V. The supplemental AC voltage can cause trapped ions of specific mass to resonate at their so-called "secular" frequency in the axial direction. When the secular frequency of an ion equals the frequency of the supplemental voltage, energy is efficiently absorbed by the ion. When enough energy is coupled into the ions of a specific mass in this manner, they are ejected from the trap in the axial direction where they can be detected as has been described. The technique of using a supplemental dipole field to excite specific ion masses is sometimes called axial modulation. As is well known in the art, axial modulation is also frequently used to eject unwanted ions from the trap, and in connection with MS/MS experiments to cause parent ions in the trap to collide with molecules of a background buffer gas and fragment into daughter ions. This latter technique is commonly referred to as collision induced dissociation (CID). As is also well known, whether an ion will be ejected by axial modulation from the trap, or instead is merely fragmented, is largely dependent on the voltage level of the supplemental dipole voltage.
The secular frequency of an ion of a particular mass in an ion trap depends on the magnitude of the fundamental trapping voltage V. Thus, them are two ways of bringing ions of differing masses into resonance with the supplemental AC voltage: scanning the frequency of the supplemental voltage in a fixed trapping field, or varying the magnitude V of the trapping field while holding the frequency of the supplemental voltage constant. Typically, when using axial modulation to scan the contents of an ion trap, the frequency of the supplemental AC voltage is held constant and V is ramped so that ions of successively higher mass are brought into resonance and ejected. The advantage of ramping the value of V is that it is relatively simple to perform and provides better linearity than can be attained by changing the frequency of the supplemental voltage. The method of scanning the trap by using a supplemental voltage will be referred to as resonance ejection scanning.
Resonance ejection scanning of trapped ions provides better sensitivity than can be attained using the mass instability technique taught by the '884 patent and produces narrower, better defined peaks. In other words, this technique produces better overall mass resolution. Resonance ejection scanning also substantially increases the ability to analyze ions over a greater mass range.
In commercial embodiments of the ion trap using resonance ejection as a scanning technique, the frequency of the supplemental AC voltage is set at approximately one half of the frequency of the AC trapping voltage. It can be shown that the relationship of the frequency of the trapping voltage and the supplemental voltage determines the value of q.sub.z (as defined in Eq. 2 above) of ions that are at resonance. Indeed, sometimes the supplemental voltage is characterized in terms of the value of q.sub.z at which it operates.
While the most common method of analyzing the contents of an ion trap involves causing ions to sequentially leave the trap in the axial direction where they can be intercepted by an external detector, other detection methods, including in-trap detection methods are well known and may be used in connection with the present invention.
Commercially, most ion traps are sold in connection with gas chromatographs (GC's) which serve, essentially, as input filters to the ion traps. As is well known, a GC serves to separate a complex sample into its constituent compounds thereby facilitating the interpretation of mass spectra. Of course, ion trap technology is not limited to use with GC's, and other sample input sources are known. For example, with an appropriate interface, a liquid chromatograph can be used as a sample source. For some applications, no sample separation is required, and sample may be introduced directly into the ion trap.
The flow from a GC is continuous, and a modem high resolution GC produces narrow peaks, sometimes lasting only a matter of seconds. In order to obtain a mass spectra of narrow peaks, it is necessary to perform at least one complete scan of the ion trap per second. The need to perform rapid scanning of the trap adds constraints which may also affect mass resolution and reproducibility. Similar constraints exist when using the ion trap with an LC or other continuously flowing, variable sample stream.
As with most any instrument of its type, it is known that the dynamic range of an ion trap is limited, and that the most accurate and useful results are attained when the trap is filled with the optimal number of ions. Ion trap mass spectrometers are extremely susceptible to deleterious effects of space charge and ion molecule reactions. The space charge in the ion trap alters the overall trapping field, interfering with mass resolution and calibration. Moreover, space charge affects the trapping efficiency and ion molecular reactions. If too few ions are present in the trap, sensitivity is low and peaks may be overwhelmed by noise. If too many ions are present in the trap, space charge effects can significantly distort the trapping field, and peak resolution can suffer.
The prior art has addressed this problem by using a so-called automatic gain control (AGC) technique which aims to keep the total charge in the trap at a constant level. In particular, prior art AGC techniques use a fast "prescan" of the trap to estimate the charge present in the trap, and then uses this prescan to control a subsequent analytical scan. While this approach has been acceptable for many applications and experiments, the inventor has determined that it does not provide highly accurate control over the space charge in the ion trap and, thus, limits the ability to obtain very high resolution.
There is an increasing demand to provide equipment which overcomes these limitations and which is capable of providing very high resolution. This demand is especially present when performing MS/MS experiments. In such circumstances it is extremely important to control the total amount of space charge in the ion trap, as explained below.
There are several prior art AGC methods that have been used to control the space charge levels in ion traps so as to optimize the performance of the trap for various applications. These prior art methods all have in common a two-step process of conducting each sample analysis: performing a prescan to estimate the concentration of sample ions present in the trap using fixed, predetermined ionization parameters, followed by an analytical scan of the trap performed using optimized the ionization parameters, based on information obtained from the prescan. The goal of these techniques is to always store approximately the same total number of ions in the trap as the sample concentration levels change. As used herein the term prescan refers to a scan of the contents of the trap which is performed for the purpose of optimizing an ionization parameter. In a prescan, no mass spectrum for use by the spectroscopist is created. A prescan is normally performed so rapidly that meaningful mass spectral data would not be discernable due to the very poor mass resolution associated with rapid scanning. As used herein the term analytical scan refers to a scan intended to collect mass spectral data of the contents of the ion trap.
In the prior art method of Stafford, et al., (U.S. Pat. No. 5,107,109) the sample concentration in the trap is measured in a prescan by applying a short, fixed-duration electron beam to the trap to cause sample ionization, followed by a rapid measurement of the total ion content of the trap. This measurement is used to control the number of sample ions in the ion trap during the subsequent analytical scan. There is no teaching to rid the trap of any unwanted ions during either the prescan or the subsequent analytical scan.
In the prior art method of Weber-Grabau, et al., (U.S. Pat. No. 4,771,172) a fixed-duration prescan is again used, in a manner similar to the method of Stafford, et al., in conjunction with chemical ionization to measure the sample concentration in the trap prior to the analytical scan. This patent also teaches eliminating unwanted sample ions from the trap during the period in which reagent ions are created in the trap. As in Stafford, et al., both the length of time that the electron beam is turned on to ionize the reagent ions, as well as the length of time the reagent ions are allowed to react with the sample to ionize it, are fixed.
The prior art method of Kelley (U.S. Pat. No. 5,200,613) also discloses a prescan which uses a short, fixed ionization time as in the method of Stafford, et al., with the improvement being the additional step of applying notched-filtered noise to the trap to resonantly eject undesired ions. The ion ejection, by means of filtered noise, to isolate parent ions, is performed in connection with both the prescan and the analytical scan. Kelley also teaches use of this process with MS/MS experiments.
All of these prior art methods suffer from utilizing fixed, predetermined ionization parameters during the prescan step to estimate the sample concentration in the trap and to adjust an ionization parameter during the subsequent analytical scan. However, a variety of ion-molecule reactions can occur within the ion trap which alter the ion intensity of a particular ion of interest, such as the parent ion in a MS/MS experiment. These processes are functions of the level (or number) of ions that are in the trap, as well as the sample concentration level that is present. The use of a fixed ionizing condition for the prescan will produce a variable number of ions, depending on how much sample is present, relative to the matrix. As will be understood by those skilled in the art, the term "matrix" includes, e.g., those molecules eluting from the GC at any given which are different from the sample compound(s) of interest. Such background molecules may be present for a variety of reasons.
The method of the '109 patent has the additional limitation in that the prescan measures the integrated ion signal from a broad mass range of ions that are trapped during the ionization period of the prescan. In a complex matrix eluting from a GC the ratio of sample to matrix can change dramatically during the elution of a sample peak from the chromatograph. Fixed ionization conditions during the prescan may increase the error in the sample level determination by including undesired ions from the matrix. Ionization of the matrix will often produce large numbers of ions with masses below that of the parent ion. Low mass ions in particular are troublesome in an ion trap, because they decrease the trapping efficiency of the higher mass parent ions. When very high concentration levels of the matrix are present, use of a fixed prescan may cause the number of sample ions that are trapped to change with the level of the matrix, even if the sample level is constant.
The method of Kelley attempts to reduce the sample/matrix problem by improving upon the method of the '109 patent by adding the additional step of applying notched filtered noise to the trap during ionization to eject unwanted ions and to isolate the parent ion. This method has the limitation of applying the notched filtered noise field to the trap during the ionization period, when the RF trapping voltage is set at a relatively low level in order to trap a broad range of masses. At low RF trapping voltages the resonance line widths of adjacent high mass ions overlap so that even the narrow frequency notches disclosed in the Kelley patent, (e.g., 1 kHz), would trap ions over range of several masses. For example, a 12-15 mass unit range would fall within a 1 kHz frequency notch at mass 400. The same notched filtered noise is used to both eject unwanted ions during the ionization period and to isolate parent ions for subsequent dissociation in an MS/MS experiment. Used in this way, notched filtered noise is non-optimum for both ion ejection and ion isolation since they are done simultaneously. Moreover, because of the continuous frequency distribution of noise, large power levels are required in order to have enough power at the secular frequency of all unwanted ions in order to eject them completely. This will result in power broadening of the ion resonance. If the notch width is made smaller to improve the resolution of the ion isolation of the parent ion, the result will be a dramatic loss in parent ion storage. This is because the line width under the trapping conditions taught by Kelley is approximately 1.5 kHz, i.e., a given ion of interest will be resonated by all frequencies within a band of frequencies 1.5 kHz wide. Under these conditions high resolution trapping is not possible.
An alternate embodiment of the method of the Kelley patent applies to MS/MS processes wherein the prescan includes the step of parent ion dissociation to form daughter ions and the subsequent integration of the daughter ion signal as a means of determining the optimizing parameters for the analytical scan. A limitation in the use of daughter ions is that the formation of daughter ions and the reproducibility of the daughter ion spectra depends on, among other factors, parent ion level and the conversion efficiency from parent to daughter ions. Thus, one of the parameters that is most affected by changes in sample level and space charge levels in the trap is the one selected by Kelley to use in the determination of the ionization parameters for the analytical scan. Moreover, this is a particular problem when using a relatively short, fixed ionization period since the relative number of daughter ions that are produced will be low, such that minor variations could cause large variations in the calculated optimum ionization time.
The general limitations of the prior art techniques are: (1) the inability to isolate only the parent ion during a prescan; (2) the inability to selectively and reproducibly store only the parent ion at a constant level as the sample and matrix levels change during a prescan; (3) when using prescans with fixed ionization conditions, the space charge conditions of the prescan will change with the sample/matrix ratio, which will affect the mass calibration for a high resolution ion isolation step, such as described in the '665 patent, as well as the extent of undesired ion-molecule reactions that occur in the trap; and (4) the estimate of the sample concentration and the determination of the optimizing parameters will be in error, as the result of an inaccurate measure of the number of ions in the trap during the prescan.